PROJECT SUMMARY This application builds upon the expertise in my laboratory to develop and apply foundational methods for analyzing and engineering cell signaling in human health. The overall vision of our research program is to create tools that enable more detailed mechanistic understanding of signaling processes that can further facilitate the design of more effective therapies. The broad goals for the next five years are to develop tools that will provide novel insights into the molecular mechanisms of receptor-ligand interactions and to analyze signaling dynamics that arise when conflicting receptor signals are simultaneously activated. In receptor biology, there is an implicit bias in what receptors are biochemically characterized: only those proteins that can be stably expressed in functional form are amenable to established biochemical assays that are used to identify the molecular basis for interaction with ligands. However, there are major classes of receptors that are biologically and clinically important but have proven highly recalcitrant in their heterologous expression. We propose to develop alternative methodologies to overcome conventional expression challenges, facilitate paratope mapping, and enable receptor re-engineering to alter ligand specificity. In ligand biology, there are established high-throughput methods for parsing the contributions of individual amino acids to receptor binding but not corresponding approaches for determining whether amino acids contribute to signal transmission. We propose to develop a methodologies that will enable mapping of a signaling ?hot spot? on a ligand and enable rapid engineering of novel agonists and antagonists. Finally, the initiation of signals via cell-surface receptors must be viewed through a holistic lens in order to understand the ultimate effect on cell response. This is most effectively illustrated by considering the simultaneous application of two ligands that, individually, drive mutually exclusive outcomes on a given cell, which is an underappreciated, but common, problem that cells in the body most resolve. Through a combination of quantitative experiments and computational modeling, this cellular ?conflict resolution? will be analyzed, providing new insights into the robust functioning of signal transduction networks. Collectively, advances in all of these areas will provide essential new tools for answering fundamental biological questions relevant to human health while also facilitating the design of novel molecular and cellular therapeutics for the treatment of a range of diseases.